The effect of substrate characteristics on primary human bone cell response to mechanical loading was investigated in this study. The substrates comprised organic and inorganic materials with a range of hydrophilic and hydrophobic features. Substrate surface topography varied from smooth to particulate to porous. It was found that hydrophilic substrates such as borosilicate glass facilitated bone cell adhesion, in contrast to hydrophobic substrates such as poly(L-lactic acid), in which clumps of cells grew unevenly across the substrate surface. All primary bone cells cultured in the various collagen-coated substrates were responsive to mechanical stimulation. The study showed that, at a low strain level of 1000 microstrain, mechanical stimulation enhanced bone cell differentiation rather than proliferation. Coating the substrates with collagen type I enhanced cell adhesion and promoted an elongated cell morphology, indicating that the presence of specific binding sites on a substrate may be more important than its hydrophilic properties, regardless of the substrate topography.